1. Field of the Invention
The present invention relates generally to the fields of cytokine biology and bone diseases. More specifically, the present invention provides inhibitors of receptor activator of NF-κB (RANK) for therapy of disorders such as diseases associated with bone resorption.
2. Description of the Related Art
Members of the TNF and TNF receptor superfamilies play critical roles in the initiation and regulation of the immune response (1-3). Although members of these families share many overlapping biological functions, it appears from gene knockout studies that they have unique features. One such receptor/ligand pair, receptor activator of NF-κB (RANK) and its ligand (RANKL/TRANCE/ODF/OPGL), is critically involved in regulation of bone remodeling and osteoclastogenesis (4). From knockout gene studies in mice, RANKL also functions in lymph node organogenesis and lymphocyte development (5). Furthermore, receptor activator of NF-κB and RANKL are implicated in the interactions between T cells and dendritic cells during the immune response (6).
As indicated by its name, receptor activator of NF-κB stimulates activation of nuclear factor-κB (NF-κB) (7-12), a transcription factor that regulates the expression of a large number of genes that play essential roles in immune and inflammatory responses (13). Evidence over the past several years has indicated that some of the TNF receptor family members interact with a family of adapter proteins known as TRAFs, (TNF receptor-associated factors), which participate in activation of the transcription factor NF-κB and c-Jun N-terminal kinase (JNK) (14).
The TNF receptor-associated factor family consists of six distinct proteins, each of which possesses a C-terminal homologous domain that is critical for self-association and is required for interaction with the receptors. All of the TNF receptor-associated factors, except for TRAF1 and TRAF4, also contain ring and zinc finger motifs in their N-termini, which appear to be utilized for interacting with other signaling molecules. Similar to the trimeric structure of the ligands and receptors of the TNF family, the C-terminus of the TRAF2 adapts a trimeric structure, as reported for TRAF2 in its interaction with peptides derived from TNF receptor 1 and CD40 (15, 16). This trimeric structure of the TNF receptor-associated factor molecules likely enables them to associate with downstream adapter proteins.
It has been demonstrated that TRAF2, TRAF5, and TRAF6 interact with receptor activator of NF-κB (RANK) and that receptor activator of NF-κB could activate both the NF-κB and JNK pathways (7). Subsequently, a more detailed analysis of the interaction of these TNF receptor-associated factors with receptor activator of NF-κB was reported (8). A novel TRAF6-binding motif has been identified in RANK that is distinct from the TRAF2- and TRAF5-binding domains. A homologous TRAF6-binding motif in CD40 was described using a combinatorial peptide library approach (17). The TRAF6 binding domain in RANK was sufficient for activation of NF-κB , suggesting that TRAF2 and TRAF5 are not necessary for NF-κB activation. However, it appears that the TRAF2-binding motif is sufficient for JNK activation, although the TRAF6-binding domain could also activate JNK, albeit to a lesser extent. Additionally, NIK (NF-κB inducing kinase) was also found to be required for the activation of NF-κB by receptor activator of NF-κB. In addition to TRAF2, TRAF5, and TRAF6, it has been demonstrated that TRAF1 and TRAF3 also associate with the carboxy terminus of receptor activator of NF-κB (9-12).
The role played by each TRAF molecule in RANK signal transduction remains elusive. Dominant negative mutants of TRAF2, TRAF5, and TRAF6 have been used to evaluate their role in NF-κB activation by RANK. It appears that all of the dominant negative TRAFs differentially inhibit the activation of NF-κB induced b y overexpression of RANK in 293 cells (9, 12). However, inclusion of all dominant negative mutants of TRAF2, TRAF5, and TRAF6 did not completely eliminate the activation of NF-κB induced by RANK in 293 cells (12). Stimulation of RANK also caused the recruitment of TRAF6, which in turn recruits and activates c-Src, which appears to be responsible for activation of phosphoinositol-3-kinase and protein kinase B/AKT, a molecule potentially involved in cell survival (18).
Knockout mouse models of RANKL, RANK, and osteoprotegerin have demonstrated an essential role of these molecules in osteoclastogenesis. The biological importance of these molecules is underscored by the induction of severe osteoporosis by targeted disruption of osteoprotegerin and by the induction of osteopetrosis by targeted disruption of RANKL or by overexpression of osteoprotegerin (5, 19, 20). Thus, osteoclast formation may be attributed to the relative ratio of RANKL to osteoprotegerin in the microenvironment of bone marrow, and alterations in this balance may be a major cause of bone loss in many metabolic bone disorders.
Similar to RANKL−/− mice, targeted disruption of receptor activator of NF-κB also lead to an osteopetrotic phenotype (21, 22). Both RANK−/− and RANKL−/− mice exhibited absence of osteoclasts, indicating the essential requirement of these molecules for osteoclastogenesis. Additionally, mice lacking TRAF6 (23-25), c-Src (26), c-Fos (27), or the NF-κB subunits p50/p52 (28, 29) also display an osteopetrotic phenotype. Although these mutant mice have osteoclasts, these cells apparently have defects in bone resorption. Thus, RANKL and receptor activator of NF-κB as well as their cytoplasmic signaling molecules are required for osteoclastogenesis.
Of the TRAF molecules that bind to receptor activator of NF-κB, only TRAF6 appears to be essential for osteoclast differentiation as indicated in mice lacking TRAF6. Thus, t h e interaction of receptor activator of NF-κB with TRAF6 may be a unique target for therapeutic intervention, and the ability to disrupt this interaction by a competitive, cell permeable peptide remains to be investigated.
Thus, the prior art is deficient in methods of disrupting the interaction between receptor activator of NF-κB and TRAF6 in order to inhibit RANKL signaling and osteoclast differentiation induced by RANKL. Such inhibitors would be useful as therapeutics in bone disorders and cancer associated with increased bone resorption. The present invention fulfills this long-standing need and desire in the art.